Nicotine imparts its effects on cellular function through interaction with neuronal nicotinic acetylcholine receptors (nAChR). Some of these receptors, especially those composed of ?4?2 subunits, respond to sustained ligand exposure through the process of upregulation as defined by a substantial increase in the density of high affinity nicotine binding sites. The physiological consequences of upregulation have been linked to many diverse processes ranging from addiction to pathophysiological processes contributing to early phases of Alzheimer's disease. An exciting and relatively new role for nAChRs is emerging with regard to the interaction with pro-inflammatory cytokines. In fact, the anti-inflammatory properties of nicotine, acting through various nAChRs, is now widely reported to influence many diseases of inflammatory etiology. However, relatively little is known about the mechanisms controlling the inflammatory:nAChR interaction. The overall goal of this proposal is to define intracellular mechanisms that regulate the interactions between key pro-inflammatory cytokines and defined combinations of nAChR subtypes. SPECIFIC AIM 1. Hypothesis: TNF?-activation of the p38MAPK pathway and/or IL-1? activation of PKA pathways enhance ?4?2 expression and these are antagonized by pathways involving PI3K/Akt signaling. In this Aim we will pursue the definition of intracellular pathways initiated by TNF? and/or IL-1? to enhance nicotine-mediated upregulation of nAChR??4?2. SPECIFIC AIM 2. Hypothesis: Direct phosphorylation of ?4 and the presence of other nAChR subunits modify the pro-inflammatory cytokine signals mediating enhanced upregulation of ?4?2. This Aim will determine the impact of: 1) modifying PKA phosphorylation sites in ?4;2) introducing (or deleting) other nAChRs (specifically ?7 or ?5);and 3) determining effects of TNF? or IL-1? intracellular signaling leading to enhanced ?4?2 upregulation in cultured neurons. SPECIFIC AIM 3. Hypothesis: Interactions between cells of the CNS such as neurons and microglia (Mg) direct the outcome of the overall inflammatory:nAChR response. Neurons and microglia will be co-cultured in different ratios to dissect the relative contribution of paracrine, autocrine or juxtacrine interactions in modulating the TNF? and IL-1?-mediated enhancement of ?4?2-upregulation in a mixed cell system. PUBLIC HEALTH RELEVANCE: We are interested in understanding the interaction between nicotine and inflammation. We will determine the signal transduction pathways cytokines use to promote or inhibit enhanced upregulation of nicotinic receptors. Two major cytokines, tumor necrosis factor alpha (TNF?) and interleukin-1? (IL-1?) enhance nicotine-induced upregulation of the high affinity nicotine receptor termed nAChR?4?2. The upregulation response is correlated with addiction behaviors and multiple pathologies of the brain such as Alzheimer's disease. Understanding how the inflammatory system and response to nicotine interact will impact upon how we approach these problems with therapeutic agents.